Clear cell variant mucoepidermoid carcinoma of salivary gland in a grey wolf (Canis lupus)

Abstract A 14‐year‐old male grey wolf (Canis lupus) with a history of severe facial swelling was submitted for necropsy. Clinical and radiological examination demonstrated an expansile neoplastic mass in the nasal and frontal sinuses. On necropsy, an amorphous neoplastic mass and extensive necrosis were observed in the nasal turbinate. Microscopic examination revealed a tumour principally composed of obvious clear tumour cells characterised by small hyperchromatic nuclei and abundant clear cytoplasm. These clear cells were positive for mucin with PAS, PAS‐D reaction, and alcian blue (pH 2.5) staining, but negative for PTAH staining. Immunohistochemically, some of tumour cells were strongly positive for mesenchymal cells (vimentin), whereas they were negative for myoepithelial antigen (alpha‐SMA) and cytokeratin. Based on the histopathological and immunohistochemical features, the present case was diagnosed as high‐grade clear cell variant mucoepidermoid carcinoma (MEC). This is the first description of clear cell variant MEC in a wolf.

Mucoepidermoid carcinoma (MEC) is the most common type of malignant epithelial salivary gland tumour (Terada et al., 2004;Varma et al., 2012). In veterinary field, the MEC has been reported in lion (Dorso et al., 2008), Amazon parrot (Nau et al., 2017), Wistar rat (Nolte et al., 1995), goat (Turk et al., 1984), dog and cat (Hammer et al., 2001). Microscopically, MEC is composed of three cell types in varying proportions: mucus-producing cells, epidermoid (squamoid) cells F I G U R E 1 Radiological examination of mucoepidermoid carcinoma, skull, grey wolf (Canis lupus). (a) Lateral radiography of the skull. Extensive osteoblastic and osteolytic changes of the frontal bones and maxillary sinuses were observed. (b) Dorsal ventral radiography of the skull. The vomer bone detail had disappeared completely. and undifferentiated intermediate cells (Ettl et al., 2012;Schwarz et al., 2011). In addition to these essential cell types, clear cells are represented in varying proportions in MEC, and some cases have been reported that clear cells infrequently predominate over other cell types (Yoon et al., 2005). Such cases are called clear cell variant MEC (Ellis, 1998). Here we report a rare case of MEC composed of abundant clear cells in a grey wolf (Canis lupus).
This case and procedures were reviewed and approved by the Animal Ethics Committee of Jeonbuk National University.
A 14-year-old male grey wolf died at the Jeonju Zoo and was referred to the Jeonbuk Veterinary Diagnostic Center for necropsy.
The wolf had a five-month history of severe facial swelling, epistaxis and purulent nasal discharge. The veterinarian of the zoo first suspected an infection of the respiratory system. After that, antibiotics and analgesics were prescribed, and the prognosis was observed.
Grossly, the wolf was emaciated, and the normal physiognomy of the craniofacial area was distorted by severe facial swelling. There were also extensive osteoblastic and osteolytic changes of the frontal bone and maxillary sinus on lateral position radiography. On dorsal ventral radiography, the vomer bone was completely obliterated, the turbinate bone and cribriform cartilage detail were lost, and there were general areas of increased soft tissue opacity in the nasal cavity ( non-neoplastic conditions such as inflammation as well as in tumours (Carberry et al., 1988). Because salivary tumour tissue has various cytological features, it is difficult to classify primary salivary gland tumours.
MEC is categorised into two basic types: 'classical' and 'variant' .
While classical MEC is composed of clearly recognisable mucous, epi-

dermoid and intermediate cells in variable proportions, variant MEC
shows the presence of ≥80% non-classical cell types, including clear cell, eosinophilic or squamoid cells (Ettl et al., 2012;Schwarz et al., 2011). Generally, clear cells account for approximately 10% of the cell population of MEC (Ellis, 1998), and they have a characteristic clear cytoplasm due to cytoplasmic accumulation of non-staining components such as glycogen, lipid, or mucins (Terada et al., 2004). The  (Fletcher, 2007). Therefore, histopathological examination is an important aspect of the diagnosis of these clear cell tumours. H&E staining is the gold standard method used for diagnosing salivary gland tumours, and various staining methods improve the accuracy of such analysis (Nagao et al., 2012) Furthermore, higher degrees of atypia, anaplasia, increased mitotic rate, necrosis, perineural and lymphovascular invasion are easily identified. Although the origin of the present tumours was unclear due to severe infiltration and extension into the surrounding tissue structures, based on the histopathological features, the wolf was diagnosed with high-grade clear cell variant MEC, and we assumed that this tumour originated from the parotid gland. This type of tumour has been reported in some domestic species and laboratory animals (Hammer et al., 2001;Ishikawa et al., 1998;Nau et al., 2017). In wildlife, however, salivary gland tumours have rarely been reported. Only one case of high-grade MEC in wildlife was previously described in the veterinary literature, in a male lion (Panthera leo) (Dorso et al., 2008).
In the lion case, microscopic features were similar to our present study. In addition, as with our results, MEC in lion showed positive for vimentin and negative for alpha-SMA. However, in the case of the lion, CKs were positive. CKs are intermediate filaments that are used for the diagnosis and classification of epithelia (Moll et al., 1982). Previous studies used CKs to evaluate the patterns in MEC, and there is a published paper that analysed the immunoprofile of CKs in different cellular types of MEC (Azevedo et al., 2008). Azevedo et al. indicated that CK 6, 7 and 8 were mainly immunopositive in clear cell type of human MEC cases. Our CKs antibody is a cocktail of anti-CK antibodies, AE1 and AE3(MNF-116, Dako, CA, USA). It has broad spectrum of reactivity against various sized and types of CK. However, in our case, cocktail CKs did not work. As the antibody's reactivity is not confirmed in all species, we assume that our antibody did not work in wolf species. Therefore, it is difficult to conclude that the clear cell variant mucoepidermoid carcinoma in wolf is negative for CKs.
In this regard, we suggest the application of CK 6, 7, and 8 antibodies that show strong reactivity in clear cell type rather than cocktail antibodies.
At five months after showing the first clinical sign, the wolf died. Metastasis to other organs and regional lymph nodes was not observed. The difficulty to eat or loss of appetite due to tumour and facial oedema may result in nutritional disorders (Solheim et al., 2014). Or in this case, as the tumour induced local osteolysis, it may be progressed to tumour-related hypercalcemia (Goldner, 2016). Also decreased immunity and secreted cytokines due to tumours may be the cause of death (Gonzalez et al., 2018).
The veterinarian in charge suspected an infection, prescribed only antibiotics and analgesics and observed the prognosis. Until the wolf died, the veterinarian did not suspect a tumour, and as the facial swelling was progressed, the veterinarian regarded the condition as an abscess arising from local infection. According to the Wildlife Protection And Management Act, Act On The Management Of Zoos And Aquariums, Animal Protection Act in the Republic of Korea, the veterinarian did not neglect animals suffering a disease. According to the results of the pathological examinations, it was confirmed that it was a highly malignant tumour. Furthermore, zoo veterinarian could not make the accurate diagnosis until the death of the animal. A good prognosis could not be expected because the wolf was an old (14-yearold) along with a malignant tumour, but if the veterinarians suspected the tumour at early stage, they would have focused on reducing the pain. Animals kept in zoos have a longer average life expectancy compared to those in wild conditions because they do not struggle for survival and are constantly monitored and treated (Müller et al., 2010).
In addition, the incidence and diagnosis rate of tumours are increasing due to prolonged lifespan and frequent medical monitoring (Hubbard et al., 1983). Therefore, this case represents a great opportunity to learn more on tumours in zoo and captive wildlife, and to promote animal welfare through practicing timely and accurate diagnosis with appropriate treatment plans.
Besides, data on tumours in wolf are limited, and until now, a squamous cell carcinoma of the tonsil and pulmonary neuroendocrine tumour were reported (Shiraki et al., 2017;Teifke et al., 2005). In this regard, our present case represents a valuable addition to the knowledge on neoplasia in wolf.
In summary, we reported an extremely rare case of high-grade clear cell variant MEC in a wolf. This is the first report in a wolf in the veterinary literature.

CONFLICT OF INTEREST
The authors declare no conflict of interest with respect to the publication of this manuscript.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHIC STATEMENT
This case and procedures were reviewed and approved by the Animal Ethics Committee of Jeonbuk National University.